Induced pluripotent stem cells: real stem cells?

The discovery of induced pluripotent stem cells (iPSCs) has provided a method for utilizing stem cell technology without the ethical controversy surrounding traditional embryonic stem cells (ESCs). Previously, consensus was that iPSCs were nearly identical to ESCs both genetically and functionally - providing hope for the translation of this technology into patient specific regenerative medicine. Recently, multiple labs have reported observations of significant genetic and epigenetic variation between iPSCs and ESCs. Further investigation via pathway based PCR analysis will be critical to determining whether iPSCs will truly be the answer to personalized regenerative medicine.

Highlights

iPSCs differ significantly from ESCs at the genomic and epigenomic level

Mouse derived ESCs were first described in the 1970's, with human stem cells reported about 20 years later (1, 2). Embryonic stem cells are derived from the inner cell mass of a fertilized egg. They exhibit pluripotency when injected into a blastocyst, meaning they have the potential to develop into all three germ layers - the endoderm, ectoderm and mesoderm - and the germ line of the resulting chimeras. ESCs express genes required for pluripotency (Oct4, Sox2, and Nanog) and females have an X chromosome that has not been inactivated. The genome of ESCs exists in a highly methylated state and the cells have the potential for unlimited proliferation (3).

Certain signaling pathways are required to maintain the pluripotent state of ESCs. Investigators identified leukemia inhibitory factor (LIF) as critical growth factor for maintaining pluripotency. They later learned that LIF maintains ESC pluripotency via Stat3 signaling. Bone morphogenetic factor 4 (BMP4) was then discovered as another growth factor that works in concert with LIF to inhibit differentiation by inducing the inhibitors of differentiation (Id) genes. Inhibition of MAPK and GSK3 signaling pathways, and thus the WNT pathway, with small molecule inhibitors was shown to inhibit differentiation as well (3, 4).

ESCs have the potential to be differentiated into nearly any type of cell, including hematopoietic cells, cardiomyocytes, oligodendrocytes and pancreatic beta cells. These cells have the potential for use in regenerative medicine.

iPSCs are produced by reprogramming somatic cells through the introduction of genes or gene products required for pluripotency. An additional benefit of this technology is that iPSCs can be developed from patient somatic cells, increasing clinical application, and decreasing the risk of negative side effects such as rejection. Induced pluripotency has developed through three generations of nuclear reprogramming: 1. nuclear transfer (also known as cloning), 2. cell fusion and 3. transcription factor transduction. In 1952, nuclei from blatocysts were transferred to enucleated Rana pipiens eggs (5). The resulting embryos developed into normal hatching tadpoles. This method of pluripotent stem cell production, known as nuclear transfer, is technically demanding in humans due to the size of the nucleus.

In 1976 investigators produced the second generation of pluripotent stem cells through fusion of thymocytes and embryonic carcinoma cells (6). The resulting chimera formed teratomas containing a wide range of differentiated tissues when transplanted into nude mice, a classical method for evaluating pluripotency. Modern technology allows us to further evaluate how closely these pluripotent stem cells resemble ESC. Fusion of somatic cells with ESC resulted in chimera exhibiting ESC-like epigenetic modification in the promoter regions of several important genes and histone acetylation and methylation (7, 8). These observations suggested that ESCs contain certain factors required for pluripotency that are absent from somatic cells.

In 2006, scientists at Kyoto University combined four transcription factors, Oct 3/4, Sox2, c-Myc, and KLF-4 with selection for Fbx15 expression and generated the first transcription factor reprogrammed stem cells (9). These cells, however did not generate adult chimeras, and thus were not bona fide iPSCs according to accepted criteria (10). Further analysis of Oct4 and Nanog promoters revealed an epigenetic pattern that was a combination of what is seen in somatic cells and ESCs, suggesting incomplete reprogramming. Less than one year later, three independent labs established adult chimera competent iPSCs through expression of the same transcription factors, but selection for Nanog rather than Fbx15 (11-13) (see figure Generation and differentiation of iPSCs). The resultant iPSC lines were believed to be nearly indistinguishable from ESCs, via analysis of global gene expression, DNA methylation, histone modification and X chromosome reactivation.

Over the last year several groups have reported genetic and epigenetic variation among ESC and iPSC lines. The differences observed fall into the categories of gene copy number variation, chromosome duplication, epigenetic variation, and acquired protein coding point mutations (see figure Genomic and epigenomic variation in iPSCs). These observations have raised a number of questions regarding functional relevance and patient safety in translation of this technology into clinical applications.

Hussein et al observed increased copy number variation in low passage iPSCs. This copy number variation appeared to have a selective disadvantage as higher passage number cell lines began to look more like ESCs (14). Laurant et al used high resolution single nucleotide polymorphism analysis to evaluate genomic stability in 186 pluripotent and 119 non-pluripotent cell line samples. They observed increased subchromosomal copy number variation in pluripotent cells; increased duplications in ESCs and deletions in iPSCs. In iPSCs, reprogramming was associated with tumor suppressor gene deletion, and increased time in culture was associated with oncogene duplication. Additional duplications arose during differentiation (15). The observations of tumor suppressor deletion and oncogene duplication in culture are of concern in translating this technology to clinical applications.
Mayshar et al analyzed the chromosomal integrity of 66 iPSCs and 38 ESCs and identified aneuploidy in early passage iPSCs. Chromosome 12 was found to be duplicated, resulting in enrichment for cell cycle related genes. This chromosome duplication may increase the tumorigenicity of these cell lines - a significant concern for clinical application (16).

Gore et al found reprogramming associated mutations enriched in cancer associated genes. While some of these mutations originated in the fibroblast progenitors, some occurred during or after reprogramming (17).
Lister et al evaluated epigenomic status in iPSCs, ESCs, somatic cells, and differentiated iPSCs and ESCs and found significant variability in reprogramming of methylation. Megabase sized regions were differentially methylated, non-CG methylation was incompletely reprogrammed, and there were differences in CG methylation and histone modification. Some of these reprogramming "errors" were found to be transmitted to differentiated cells (18).

These observations of genomic and epigenomic aberrations, often involving cancer related genes, highlight the importance of careful monitoring of the iPSCs genomic state. A number of questions remain regarding whether such genomic events have any effect on the function, stability, differentiation potential, or safety of iPSCs. What causes these genetic and epigenetic alterations? Some originate in the somatic cells but some are arising during reprogramming or from culture conditions. How can we determine harmless from deleterious alternations? Some alterations are self limiting and can be selected out through culture; some alterations appear to be promoter through culture conditions. Can we develop iPSCs that more closely resemble ESCs? Does it matter? These recent reports highlight the need for tools for rapid profiling and analysis of iPSC lines.

Development of an iPS cell line requires reprogramming with a cocktail of transcription factors. Then the cell lines need to be tested for pluripotency by evaluating the expression and methylation status of a number of genes. Once cells are differentiated they can be evaluated for expression of signature associated genes.

Laurent, L. C., et al., (2011) Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell. Stem. Cell. 8, 106–18.